靶向NS3蛋白的双抗夹心ELISA检测牛病毒性腹泻病毒OA

In order to achieve convenient and large-scale detection of bovine viral diarrhea virus(BVDV)NS3 protein,monoclonal antibodies against NS3 protein were prepared and identified,purified and horseradish peroxidase(HRP)-labeled,then rabbit polyclonal antibodies against NS3 protein were used as the coated capture antibodies and the HRP-labeled monoclonal antibodies were used as the detection antibodies.After a series of condition optimizations and screenings,a BVDV sandwich ELISA antigen detection method was established.The specificity,sensitivity and repeatability of this detection method were evaluated.This detection method has been applied to clinical sample testing,and the test results have been used to analyze the coincidence rate with commercial ELISA kits.Results were as follows:Through the screening and identification of the prepared monoclonal antibody,a monoclonal antibody 4B3 with excellent reactivity was selected for purification and HRP-labeling.The optimized reaction conditions of the sandwich ELISA are as follows:the coating concentration of the capture antibody is 2 μg·mL-1;the dilution of the sample to be tested is 1∶2,and it is incubated at 37℃for 45 minutes;the dilution of the detection antibody is 1∶2 000,and it is incubated at 37℃for 45 minutes;color development at 37℃in the dark for 15 minutes.This method was used to test 200 negative samples,and the cut-off value was determined to be 0.154 6.This method has no cross-reaction against CSFV,IBRV,BPIV-3,Mycoplasma bovis.The sensitivity test results show that the minimum detection limit of this method for different subtypes of virus samples is 103.29 TCID50·mL-1,and the minimum detection limit for NS3 protein is 0.007 8 μg·mL-1.The intra-batch and inter-batch CV%of this method are both lower than 10%.The 601 clinical serum and ear tissue samples were detected,the total coincidence rate of the test results with the commercial ELISA kits was 97.7%.The sandwich ELISA antigen detection method established in this study has good specificity,sensitivity and repeatability.The establishment of this method provides a reliable technical approach for rapid screening of infected animals.